Methods and reagents for characterizing thsd1 function

ABSTRACT

The disclosure describes global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown and identification from these studies of specific genes and pathways that are THSD1. The analysis highlights a role for THSD1 in regulating endothelial to mesenchymal transition and other pathways. The analysis also demonstrates that endothelial cell injury is the first event of an intracranial aneurysm. The disclosure provides novel cell lines and reagents used for better understanding the molecular mechanism of THSD1 biology.

CROSS-REFERENCE

The present application claims priority to U.S. Provisional Application Ser. No. 63/296,817, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,820, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,821, filed Jan. 5, 2022; and U.S. Provisional Application Ser. No. 63/296,825, filed Jan. 5, 2022, the contents of each being hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to the characterization of molecular targets for treating intracranial aneurysms (IA). Intracranial aneurysm (IA) is a cerebrovascular disease that predominantly occurs in the cerebral artery and is characterized by pathologic dilatation of blood vessels. Each intracranial aneurysm (IA) is a weakened area in a cerebral artery wall that leads to abnormal dilatation and rupture causing subarachnoid hemorrhage (SAH), a major cause of hemorrhagic stroke. A rupture of IA induces a subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke that frequently leads to death or severe disability. Due to early age of onset and high mortality, SAH accounts for >25% of years lost for all stroke victims under the age of 65 years. Despite treatment advances, SAH mortality rate is 40% and only half of survivors return to independent life.

There is a critical unmet need for understanding the genetic and molecular basis for IA to improve clinical outcomes through early therapeutic intervention.

SUMMARY

This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

In some aspects provided herein are a cell line(s) comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances, the THSD1 allele codes for an isoform of THSD1. The isoform of THSD1 can be encoded by a single codon substitution compared to the wild type allele, such as a single codon substitution that provides a missense mutation compared to the wild type THSD1 protein. The single codon substitution in the THSD1 allele can be, for example, at codon 5, at codon 460, at codon 466, at codon 600, at codon 639, at codon 653, or at codon 775. Such single codon substitution can lead to the following missense mutations, for example, a Leucine for a Phenylalanine (L5F) substitution, an Arginine for a Tryptophan (R460W) substitution, a Glutamic Acid for a Glycine (E466G) substitution, a Glycine for a Glutamic Acid (G600E) substitution, a Proline for a Leucine (P639L) substitution, a Threonine for a Isoleucine (T653I) substitution, a Serine for a Proline (S775P) substitution, or any combination thereof. In some instances the cell line is human and in other instances is murine. In some instances, the cell line is derived from a human endothelial cell, which can be from a brain tissue or from umbilical vein endothelial cells. In some instances, the human endothelial cell is immortalized from a primary cell.

In some aspects provided herein is a chimeric nucleic acid molecule, comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene. In some instances, the chimeric nucleic acid molecule further comprises a drug selection cassette. Such chimeric nucleic acid molecules may further comprise a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances, the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest. In some instances the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In some instances, such single codon substitution can lead to the following missense mutations, for example, a Leucine for a Phenylalanine (L5F) substitution, an Arginine for a Tryptophan (R460W) substitution, a Glutamic Acid for a Glycine (E466G) substitution, a Glycine for a Glutamic Acid (G600E) substitution, a Proline for a Leucine (P639L) substitution, a Threonine for a Isoleucine (T653I) substitution, a Serine for a Proline (S775P) substitution, or any combination thereof.

In some aspects the disclosure provides a method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof. In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii). In some instances, the heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof. In some instances, the non-human animal is a rat or a mouse.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A (FIG. 1A) illustrates simplified pedigrees of three IA families (NR4748, NR4931, and NR5704). Specifically, FIG. 1A illustrates Intracranial Aneurysm (IA) affected (black), aortic aneurysm (AA) affected (half black), unaffected individuals >45 (white), and individuals with unknown/unclear status (gray symbols).

FIG. 1B (FIG. 1B) depicts patient-identified THSD1 variants identified in the pedigrees of the families from FIG. 1B. The black bar represents the 853 amino acids of the WT version of the THSD1 gene. The diagram lists 8 variants found in patients affected by IA and AA, namely LSF, R450X, R460W, E466G, G600E, P639L, T653I, S775P. The back boxes illustrate the relative position of the signal peptide (SP), thrombospondin type 1 domain (TSP1), and transmembrane (TM) domains of THSD1.

FIG. 2 (FIG. 2 ) is a drawing depicting an intracranial aneurysm. An intracranial aneurysm, also known as a brain aneurysm, is a cerebrovascular disorder in which weakness in the wall of a cerebral artery or vein causes a localized dilation or ballooning of the blood vessel.

FIG. 3 (FIG. 3 ) is depicts RNAseq data for two THSD1 knock-down endothelial cell lines. Both cell lines support the evidence that THSD1 mediates endothelial to mesenchymal transition (EndMT) function (THSD1 Knock-down induces the activations of EMT—loss of endothelial normal function, but increased inflammatory cell).

FIG. 4 (FIG. 4 ) is a western blot illustrating the generation of human cell lines expressing patient-identified THSD1 variants LSF, R460W, E466G, G600E, P639L, T6521, and S775P.

FIG. 5 (FIG. 5 ) is a protein alignment between human and zebrafish THSD1.

FIG. 6 (FIG. 6 ) is a sequence alignment illustrating three rare variants of thsd1 conserved between humans and fish.

It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTIONS

In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.

The rupture of an intracranial aneurysm frequently causes a subarachnoid hemorrhage (SAH), a type of stroke characterized by high morbidity and mortality. Specifically, the present disclosure demonstrates with data from three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status.

Previously, it has been reported that deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants caused disease in both familial and sporadic cases with supporting evidence from animal models. Of note, whole exome sequencing of large IA families identified (some members of the affected family are shown in the pedigree of FIG. 1A) a THSD1 nonsense mutation that segregated in all nine (9) affected and was absent in 13 unaffected family members. It was further discovered that eight (8) THSD1 rare missense variants in 507 unrelated patients/probands where each perturbed THSD1 cell adhesion activity.

These rare variants were highly enriched in case-control studies in comparison to ethnically matched controls. It was found that Thsd1 loss-of-function leads to brain hemorrhage and premature death in both zebrafish and mice. Further, Thsd1 heterozygous and null mice developed IA and suffered SAH. The study further demonstrated that THSD1 is highly expressed in endothelial cells of the cerebrovasculature, is important for cell adhesion, promotes nascent focal adhesion assembly via Talin interactions, and potentially regulates downstream signaling. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference in their entireties). However the study did not provide any insights on the THSD1 molecular pathways.

We hypothesized that other high-risk rare genetic variants could cause IA and their identification could provide critical insights into disease pathobiology, however numerous challenges exist in identifying and characterizing genes whose perturbation leads to intracranial aneurysms (IA) and/or vascular endothelial cell dysfunction. Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants. Yet, these studies are frequently limited by small family pedigrees available for study and usually do not provide a sufficient number of repeats to be statistically significant.

To further study the role of THSD1 in IA/SAH, additional analysis of whole exome sequencing of the IA families described in FIG. 1 was conducted. The detailed analysis identified a THSD1 nonsense mutation that segregated in all 9 affected and was absent in 13 unaffected family members. Notably, the exome sequencing analysis uncovered multiple signaling pathways that appeared to be affected by THSD1 expression: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling as well as TGFβ signaling (see Table 1, discussed in Example 1). The present disclosure contemplates that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression.

The present disclosure considered the differentially expressed genes and characterized autophagy pathways as contributors to IA development and potential targets for therapy. The present disclosure also contemplates that mutations in genes other than THSD1 that affect the TGFβ pathway could render a subject at risk of suffering an IA. The present disclosure characterizes the TGFβ pathway as a novel molecular target for the treatment of subjects at risk of developing an aneurysm.

We subsequently used whole exome sequencing and discovered that THSD1 is linked molecularly to endothelial dysfunction mediated through the biological process of endothelial to mesenchymal transition (EndMT).

Definitions

All of the functionalities described in connection with one embodiment of the methods, devices or instruments described herein are intended to be applicable to the additional embodiments of the methods, devices and instruments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of steps, components, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.

As used in the specification and claims of this application, the term “administering” includes any method which is effective to result in delivery of an autophagy inhibitor to the subject.

As used in this specification, the term “aneurysm” refers to broad classes of aneurysm, including aneurysms: abdominal aortic, thoracic aortic, and cerebral.

As used in this specification, the term “cerebral aneurysm” or “intracranial aneurysm” (also known as a brain aneurysm) is a weak or thin spot on an artery in the brain that balloons or bulges out and fills with blood. The bulging aneurysm can put pressure on the nerves or brain tissue. It may also burst or rupture, spilling blood into the surrounding tissue (called a hemorrhage). An unruptured aneurysm usually causes no symptoms. A key symptom of a ruptured aneurysm is a sudden, severe headache. Treatments for an unruptured aneurysm include medications to control blood pressure and procedures to prevent a future rupture.

As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.

As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.

As used in this specification, the term “thoracic aortic” aneurysm is an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel. A thoracic aortic aneurysm occurs in the part of the body's largest artery (the aorta) that passes through the chest.

As used in this specification the “wild-type” or “WT” thrombospondin type 1 domain containing 1 (THSD1) sequence refers to the human sequence identified by NCBI Reference Sequence: NG 047168.1; unless otherwise stated.

As used herein a missense mutation refers to a change in one amino acid in a protein, arising from a point mutation in a single nucleotide. Missense mutation is a type of nonsynonymous substitution in a DNA sequence that is translated into a nonsynonymous substitution in a protein sequence. As used herein, missense mutations do not encompass nonsense mutations—in which a codon is changed to a premature stop codon that results in truncation of the resulting protein.

A used herein, an “immortalized cell line” is a cell line derived from cells that have been artificially manipulated to proliferate indefinitely and can, thus, be cultured over several generations.

Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

A. Cell Lines with Missense Mutations

A variety of cells and non-human animals comprising a modified THSD1 locus (e.g. THSD1 allele) are described herein. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. In many instances, the missense mutations identified and characterized in the disclosure present an autosomal dominant phenotype, and an insertion of one allele having the missense mutation is sufficient to achieve a phenotype associated with an aneurysm in a cell or in an animal.

The cells provided herein can be, for example, any non-human cell comprising an Thsd1 locus or a genomic locus homologous or orthologous to the human CACNG1 locus. The cells can be, e.g., mammalian cells, non-human mammalian cells, and human cells. The term “animal” includes mammals, fishes, and rodents. A mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, or a zebrafish cell. Other non-human mammals include, for example, any non-human animal that carries a homolog of the Thsd1 gene and is suitable for study of phenotypes associated with lack of function of the THSD1 protein. Birds include, for example, chickens, turkeys, ostrich, geese, ducks, and so forth. Domesticated animals and agricultural animals are also included. The term “non-human” excludes humans.

Primary cells reach senescence after limited generation and the process of frequently re-establishing fresh cultures from explanted tissues is onerous and often impracticable. Using immortalized primary cells or cell lines provides the ability best understand missense mutations that are not easily captured with the study of knock-down or knock-out cell lines. In notable instances (e.g., sickle cell) the missense mutation has additional properties and changes the function of the protein in a way that cannot be studied or elucidated with the mere review of function of the wild-type protein. Thus, the disclosure provides methods and strategies for studying proteins bearing missense mutations in the THSD1 gene. Primary cells can be immortalized with hTERT-based strategy in a manner that retains characteristics of the differentiated cell types with tissue-specific features, differentiation-specific proteins associated with the cells that the proteins are derived from. Further, technologies such as CRISPR can be used to create knock-in of the identified THSD1 missense mutations, namely LSF, R450X, R460W, E466G, G600E, P639L, T653I, S775P, in established immortalized cell line backgrounds.

Thus, in some aspects, suitable cells provided herein also include primary cells. Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. They include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture. Such cells can be rare, for example, endothelial cells (e.g., Human brain microvascular endothelial cells (HBMECs) cells; see Example 1), and can be isolated and immortalized to facilitate the study of a phenotype observed in the family described in Example 1.

Telomerase Reverse Transcriptase (TERT) Expression for Immortalizing Cell Lines

Telomerase is a ribonucleoprotein that can extend the DNA sequence of telomeres, which lets the cells undergo infinite cell divisions through evading the senescence process. This Telomerase Reverse Transcriptase (TERT) expression is generally inactive in most somatic cells, ibut when exogenously expressed, the cells are able to maintain suffcient telomere lengths for avoiding replicative senescence. This approach can be used for immortalizing cell lines with the identified THSD1 missense mutations, namely LSF, R450X, R460W, E466G, G600E, P639L, T653I, S775P.

Viral Gene Based Immortalization

Viral genes can affect the cell cycle by deregulating the biological brakes on the proliferative control of the cells. In some aspects, the disclosure contemplates using viral genes, such as the simian virus 40 (SV40) T-antigen for immortalization of cells; in other instances the disclosure contemplates using viral oncogenes.

The cells can also be any type of undifferentiated or differentiated state. For example, a cell can be a totipotent cell, a pluripotent cell (e.g., a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types. Such pluripotent and/or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that are capable of contributing to any tissue of the developing embryo upon introduction into an embryo. ES cells can be derived from the inner cell mass of a blastocyst and are capable of differentiating into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

B. Chimeric Nucleic Acids Encoding Thsd1 Sequences Having Missense Mutations

Also described herein is a chimeric nucleic acid molecule comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene. In some instances, the chimeric nucleic acid molecule further comprises a drug selection cassette. In some instances, the chimeric nucleic acid molecule further comprises a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele. In some instances the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest.

In specific instances, the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In more specific instances, the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; or (viii) any combination of (i)-(vii).

C. Methods of Making Non-Human Animals Comprising a Heterologous Cacng1 Locus

In some aspects, the disclosure provides a method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof.

Various methods are suitable for making a non-human animal comprising a heterologous locus. Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, e.g., Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1-19.11.22 and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is herein incorporated by reference in its entirety for all purposes. For zebra fish methodologies see, e.g., Brief Funct. Genomics. 2016 July; 15(4):322-30. “Genome editing in zebrafish: a practical overview.” See also, Curr Protoc Mol Biol. 2017 Jul. 5; 119:31.9.1-31.9.22. “CRISPR/Cas9-Directed Gene Editing for the Generation of Loss-of-Function Mutants in High-Throughput Zebrafish F0 Screens”. Such genetically modified non-human animals can be generated, for example, through gene knock-in at a targeted Thsd1 locus.

For example, the method of producing a non-human animal comprising a humanized Thsd1 locus can comprise: (1) modifying the genome of a pluripotent cell to comprise the humanized Thsd1 locus; (2) identifying or selecting the genetically modified pluripotent cell comprising the humanized Thsd1 locus; (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo cells in vitro; and (4) implanting and gestating the host embryo cells in a surrogate mother. Optionally, the host embryo comprising modified pluripotent cell (e.g., a non-human ES cell) can be incubated until the blastocyst stage before being implanted into and gestated in the surrogate mother to produce an F0 non-human animal. The surrogate mother can then produce an F0 generation non-human animal comprising the humanized Thsd1 locus.

The methods can further comprise identifying a cell or animal having a modified target genomic locus. Various methods can be used to identify cells and animals having a targeted genetic modification.

The screening step can comprise, for example, a quantitative assay for assessing modification of allele (MOA) of a parental chromosome. For example, the quantitative assay can be carried out via a quantitative PCR, such as a real-time PCR (qPCR). The real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes a non-targeted reference locus. The primer set can comprise a fluorescent probe that recognizes the amplified sequence.

Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655, incorporated herein by reference in its entirety for all purposes).

An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cell can be generated, for example, through recombination by (a) introducing into the cell one or more targeting vectors comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites, wherein the insert nucleic acid comprises a heterologous Thsd1 locus; and (b) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the target genomic locus. Alternatively, the modified pluripotent cell can be generated by (a) introducing into the cell: (i) a nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site within the target genomic locus; and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5′ and 3′ homology arms corresponding to 5′ and 3′ target sites located in sufficient proximity to the recognition site, wherein the insert nucleic acid comprises the heterologous Thsd1 locus; and (c) identifying at least one cell comprising a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used. Examples of suitable nucleases include a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems or components of such systems (e.g., CRISPR/Cas9). See, e.g., US 2013/0309670 and US 2015/0159175, each of which is herein incorporated by reference in its entirety for all purposes.

The donor cell can be introduced into a host embryo at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4 cell stage or the 8 cell stage). Progeny that are capable of transmitting the genetic modification though the germline are generated. See, e.g., U.S. Pat. No. 7,294,754, herein incorporated by reference in its entirety for all purposes.

In some instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii). In specific instances, the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii). In specific instances, the non-human animal comprises a heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof.

The non-human animal can be a rat, a mouse, a zebrafish, or any suitable animal having an ortholog of the human Thsd1 gene.

EMBODIMENTS

EMBODIMENT 1. A cell line comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.

EMBODIMENT 2. The cell line of embodiment 1, wherein the THSD1 allele codes for an isoform of THSD1.

EMBODIMENT 3. The cell line of embodiment 2, wherein the isoform of THSD1 is encoded by a single codon substitution compared to the wild type allele.

EMBODIMENT 4. The cell line of embodiment 2, wherein the isoform of THSD1 carries a missense mutation compared to the wild type THSD1 protein.

EMBODIMENT 5. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 5.

EMBODIMENT 6. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 460.

EMBODIMENT 7. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 466.

EMBODIMENT 8. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 600.

EMBODIMENT 9. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 639.

EMBODIMENT 10. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 653.

EMBODIMENT 11. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele is at codon 775.

EMBODIMENT 12. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (LSF) substitution.

EMBODIMENT 13. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution.

EMBODIMENT 14. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution.

EMBODIMENT 15. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution.

EMBODIMENT 16. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution.

EMBODIMENT 17. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution.

EMBODIMENT 18. The cell line of embodiment 3, wherein the single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution.

EMBODIMENT 19. The cell line of any of embodiments 2-18 wherein the cell line is human.

EMBODIMENT 20. The cell line of embodiment 1, wherein the cell line is derived from a human endothelial cell.

EMBODIMENT 21. The cell line of embodiment 20, wherein the human endothelial cell is derived from a brain tissue.

EMBODIMENT 22. The cell line of embodiment 20, wherein the human endothelial cell is derived from umbilical vein endothelial cells.

EMBODIMENT 23. The cell line of embodiment 20, wherein the human endothelial cell is immortalized from a primary cell.

EMBODIMENT 24. A chimeric nucleic acid molecule, comprising a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein and (b) is modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof, wherein the chimeric nucleic acid molecule encodes a missense THSD1 protein, and optionally, wherein the chimeric nucleic acid sequence further comprises promoter and/or regulatory sequences of the Thsd1 gene.

EMBODIMENT 25. The chimeric nucleic acid molecule of embodiment Error! Reference source not found., wherein the chimeric nucleic acid molecule further comprises a drug selection cassette.

EMBODIMENT 26. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found.—Error! Reference source not found., further comprising: (i) a 5′ homology arm upstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele; and (ii) a 3′ homology arm downstream of the Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.

EMBODIMENT 27. The chimeric nucleic acid molecule of embodiment Error! Reference source not found., wherein the 5′ homology arm and 3′ homology arm undergo homologous recombination with a non-human animal locus of interest, and wherein following homologous recombination with the non-human animal locus of interest, the modified Thsd1 allele replaces the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest and is operably linked to an endogenous promoter that drives expression of the non-human animal Thsd1 allele at the non-human animal Thsd1 locus of interest.

EMBODIMENT 28. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii).

EMBODIMENT 29. The chimeric nucleic acid molecule of any one of embodiments Error! Reference source not found., wherein the heterologous missense THSD1 protein or a portion thereof comprises (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).

EMBODIMENT 30. A method of making a Thrombospondin Type 1 Domain Containing 1 (THSD1) non-human animal comprising modifying an endogenous Thsd1 locus of the non-human animal to encode a heterologous THSD1 protein comprising a missense mutation or a portion thereof.

EMBODIMENT 31. The non-human animal of embodiment Error! Reference source not found., wherein the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5; (ii) a single codon substitution in the THSD1 allele at codon 460; (iii) a single codon substitution in the THSD1 allele at codon 466; (iv) a single codon substitution in the THSD1 allele at codon 600; (v) a single codon substitution in the THSD1 allele at codon 639; (vi) a single codon substitution in the THSD1 allele at codon 653; (vii) a single codon substitution in the THSD1 allele at codon 775; or (viii) any combination of (i)-(vii).

EMBODIMENT 25. The non-human animal of claim Error! Reference source not found., wherein the endogenous Thsd1 locus of the non-human animal comprises: (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).

EMBODIMENT 26. The non-human animal of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the heterologous THSD1 protein comprising the missense mutation or the portion thereof comprise an amino acid sequence of a human THSD1 protein or a portion thereof.

EMBODIMENT 27. The non-human animal of any one of embodiments Error! Reference source not found.-Error! Reference source not found., wherein the non-human animal is a rat or a mouse.

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

The practice of some molecular techniques described herein may employ, unless otherwise indicated, techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such techniques and descriptions can be found in standard laboratory manuals such as Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.; all of which are herein incorporated in their entirety by reference for all purposes.

Example 1: Identification of Novel THSD1 Variants in Human Samples

Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants. FIG. 1 illustrates a large family pedigree studied as part of the present disclosure to identify novel candidate genes whose rare variants cause intracranial aneurysm. Specifically, we studied three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease.

For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status. The detailed analysis identified a THSD1 nonsense mutation that segregated in all 9 affected and was absent in 13 unaffected family members. The following additional THSD1 missense variants were discovered in 507 unrelated patients/probands where each perturbed THSD1 cell adhesion activity. They are LSF, R460W, E466G, G600E, P639L, T653I, or S775P.

Example 2: Molecular Dissection of THSD1 Function in Vascular Endothelial Cells from Global Transcriptomics

Genetic factors play a significant role in IA pathogenesis as illustrated by family studies and several IA predisposing syndromes. 7%-20% of all patients have a known family history and a family history is the strongest risk factor for disease. Excluding syndromes that account for less than 1% of all IA cases, candidate IA genes have been primarily identified by genome-wide association studies and more recently, by whole exome sequencing in affected families. Yet, little is known about the genetic causes of IA providing minimum insight for the understanding and development of therapeutic targets that could treat the disease.

Deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants cause disease in both familial and sporadic cases with supporting evidence from animal models. THSD1 is predominantly expressed in vascular endothelial cells. The work identified deleterious variants in thrombospondin-type 1 domain-containing protein 1 (THSD1) that can cause IA and SAH. Initial characterization of Thsd1 in two vertebrate models including zebrafish and mice lead to the discovery that THSD1 mediated cerebral hemorrhage is located in subarachnoid space in mice. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference).

However, the mechanism of action utilized by the discovered THSD1 variants to drive disease remained elusive. Further, there was little information describing genes and pathways regulated by THSD1 using global transcriptomics that could be used to inform the mechanism of action of THSD1. Thus, on its own the identification of THSD1 in the context of AI was not sufficient to inform a therapeutic strategy.

The present disclosure contemplated that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression. The present disclosure provides results from global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown that identifies THSD1-regulated specific genes and pathways that are critical for mediating its function, providing potential targets for therapeutic intervention in IA.

The instant disclosure provides RNAseq experiments in two THSD1 knock-down endothelial cell lines. The RNAseq results from both cell lines support the evidence that THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling. A few of these pathways were selected for further analysis and characterization.

Materials and Methods

Cell Culture

HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.

Knock-Down Experiments

Knockdown experiments in human vascular endothelial cells were performed using two distinct cell lines [HUVECs and Human brain microvascular endothelial cells (HBMECs)] using four siRNAs (two control siRNAs and two THSD1-specific siRNAs) to minimize erroneous findings due to off-target effects.

Transcriptome Profiling

Bioinformatic analyses of the global transcriptome were performed on rRNA-depleted RNA samples by RNA-Seq. Table 1 illustrates results of the analysis. As shown on Table 1, THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling) as well as TGFβ signaling.

TABLE 1 Statistically Significant THSD1-Regulated Pathways (p-value < 0.005; Efron-Tibshirani's GSA test) Description Integrin Signaling PI3K/Akt/mTOR Signaling Src Signaling Rho Signaling Tgfβ

We identified a number of genes that are affected by the lack of THSD1 in the knock-down cell lines and are likely regulated by THSD1. A subset of these genes likely contributes to disease pathobiology and may be targets for therapeutic intervention. Table 2. Describes genes differentially expressed in THSD1 knockdowns.

TABLE 2 Genes differentially expressed in THSD1 knockdowns; human brain microvascular endothelial cells and HUVECs, combined Description Genes Differential Expression ALOX5AP, ARL17B, CTSS, GUK1, upon THSD1 Loss IFNAR2, LAMC2, NHLRC4, NISCH, Upregulated genes OLFML3, PSMD11, PUM2, SLC37A1, SMAP1, SNORA27, TMEM50B, UBAP2, ZNF780A Differential Expression ABCG2, ADAMTS1, AQP1, BCAP31, upon THSD1 Loss CDC45, CHCHD7, DMD, ENOX2, Downregulated genes FAM219A, FGF2, FGFR1OP2, G0S2, GJA4, IGFBP3, INAFM1, MAP2, MGP, NOL3, NPR1, NVL, PPHLN1, SERPINB2, SETBP1, (THSD1), TMEM107, ZNF185

Table 3. lists genes differentially expressed in THSD1 knockdown HUVECs.

TABLE 3 Differentially Expressed Genes in THSD1 Knockdown HUVECs Ensembl Gene Gene Name logFC logCPM LR PValue FDR ENSG00000136114 THSD1 −2.07 4.85 141.12 1.52E−32 2.15E−28 ENSG00000265107 GJA5 −3.81 3.42 137.13 1.13E−31 8.01E−28 ENSG00000130066 SAT1 −1.74 8.37 128.15 1.04E−29 4.92E−26 ENSG00000111341 MGP −1.46 6.50 112.09 3.42E−26 1.21E−22 ENSG00000187513 GJA4 −2.29 4.32 102.47 4.39E−24 1.25E−20 ENSG00000284057 AP001273.2 8.67 −0.99 71.34 3.00E−17 7.11E−14 ENSG00000170323 FABP4 −2.09 6.36 66.29 3.90E−16 7.91E−13 ENSG00000125266 EFNB2 −1.28 7.77 63.28 1.79E−15 3.18E−12 ENSG00000113389 NPR3 −3.16 1.25 56.87 4.65E−14 7.35E−11 ENSG00000134668 SPOCD1 1.16 5.45 54.03 1.97E−13 2.80E−10 ENSG00000118777 ABCG2 −2.38 4.92 51.45 7.36E−13 9.50E−10 ENSG00000203812 HIST2H2AA3 4.21 0.20 50.59 1.14E−12 1.35E−09 ENSG00000272921 AC005832.4 −8.85 −0.72 48.91 2.69E−12 2.93E−09 ENSG00000078018 MAP2 −1.06 4.84 48.51 3.29E−12 3.34E−09 ENSG00000115884 SDC1 −1.53 3.23 48.33 3.61E−12 3.42E−09 ENSG00000139278 GLIPR1 1.13 6.53 47.73 4.89E−12 4.34E−09 ENSG00000101335 MYL9 1.09 4.76 47.07 6.83E−12 5.71E−09 ENSG00000181634 TNFSF15 1.64 5.10 45.62 1.43E−11 1.13E−08 ENSG00000122861 PLAU 1.03 5.58 43.85 3.55E−11 2.65E−08 ENSG00000107551 RASSF4 −1.10 5.07 43.68 3.86E−11 2.74E−08 ENSG00000274611 TBC1D3 −7.97 −1.52 43.13 5.12E−11 3.46E−08 ENSG00000149591 TAGLN 1.81 2.54 42.71 6.35E−11 4.10E−08 ENSG00000171435 KSR2 1.18 3.90 38.39 5.80E−10 3.58E−07 ENSG00000153165 RGPD3 4.26 −1.48 37.47 9.26E−10 5.48E−07 ENSG00000251569 AC093899.2 7.32 −2.20 34.85 3.55E−09 2.02E−06 ENSG00000163083 INHBB −2.57 2.18 34.75 3.75E−09 2.05E−06 ENSG00000272949 AC093668.2 −7.24 −2.15 33.67 6.51E−09 3.43E−06 ENSG00000152217 SETBP1 −1.78 3.75 33.17 8.42E−09 4.27E−06 ENSG00000119900 OGFRL1 0.85 6.59 32.79 1.03E−08 5.02E−06 ENSG00000082684 SEMA5B −4.57 −1.62 32.23 1.37E−08 6.26E−06 ENSG00000169604 ANTXR1 0.98 5.91 32.24 1.36E−08 6.26E−06 ENSG00000137573 SULF1 −1.27 5.03 31.88 1.64E−08 7.29E−06 ENSG00000115232 ITGA4 1.35 3.29 30.82 2.83E−08 1.22E−05 ENSG00000058085 LAMC2 1.06 5.71 30.05 4.21E−08 1.76E−05 ENSG00000022567 SLC45A4 −1.35 3.07 29.70 5.04E−08 1.99E−05 ENSG00000172602 RND1 −1.74 1.64 29.71 5.03E−08 1.99E−05 ENSG00000103426 CORO7-PAM16 4.38 −1.48 29.02 7.16E−08 2.75E−05 ENSG00000120217 CD274 1.35 4.21 28.77 8.14E−08 3.04E−05 ENSG00000116667 C1orf21 −0.99 4.42 27.88 1.29E−07 4.70E−05 ENSG00000169129 AFAP1L2 −2.16 3.16 27.67 1.44E−07 5.10E−05 ENSG00000138646 HERC5 −1.85 1.47 27.51 1.56E−07 5.29E−05 ENSG00000197646 PDCD1LG2 1.07 3.58 27.55 1.53E−07 5.29E−05 ENSG00000064651 SLC12A2 0.73 7.05 26.91 2.13E−07 7.03E−05 ENSG00000171388 APLN 0.85 9.85 26.02 3.39E−07 0.000109 ENSG00000175426 PCSK1 1.51 2.68 25.80 3.79E−07 0.00012 ENSG00000057019 DCBLD2 0.91 6.78 25.67 4.05E−07 0.000125 ENSG00000008517 IL32 0.69 6.11 25.52 4.37E−07 0.000132 ENSG00000138685 FGF2 −1.20 5.72 25.21 5.15E−07 0.000152 ENSG00000065308 TRAM2 0.67 8.10 25.04 5.60E−07 0.000162 ENSG00000137507 LRRC32 −0.87 7.22 24.74 6.57E−07 0.000187 ENSG00000129116 PALLD 0.69 6.27 24.56 7.22E−07 0.000201 ENSG00000129521 EGLN3 −1.85 0.09 24.44 7.66E−07 0.000209 ENSG00000125954 CHURC1-FNTB −6.59 −2.67 24.25 8.46E−07 0.000227 ENSG00000152402 GUCY1A2 1.90 1.31 24.20 8.68E−07 0.000228 ENSG00000169429 CXCL8 1.22 4.62 23.66 1.15E−06 0.000297 ENSG00000164683 HEY1 −1.25 3.49 23.30 1.38E−06 0.000345 ENSG00000176907 TCIM 0.95 3.77 23.31 1.38E−06 0.000345 ENSG00000082126 MPP4 0.76 4.76 23.08 1.56E−06 0.000379 ENSG00000163637 PRICKLE2 −0.99 3.76 23.06 1.57E−06 0.000379 ENSG00000148143 ZNF462 0.72 4.95 22.92 1.69E−06 0.0004 ENSG00000139289 PHLDA1 0.63 7.00 22.22 2.43E−06 0.000566 ENSG00000028137 TNFRSF1B −0.99 6.14 22.02 2.69E−06 0.000617 ENSG00000139629 GALNT6 0.66 5.98 21.94 2.82E−06 0.000635 ENSG00000117586 TNFSF4 0.66 7.43 21.80 3.03E−06 0.000672 ENSG00000256514 AP003419.1 1.68 0.91 21.47 3.60E−06 0.000787 ENSG00000240583 AQP1 −1.84 3.43 21.27 3.99E−06 0.000858 ENSG00000115252 PDE1A −2.04 0.57 21.17 4.20E−06 0.00089 ENSG00000178695 KCTD12 −0.71 8.94 20.99 4.62E−06 0.000965 ENSG00000173535 TNFRSF10C −0.65 6.69 20.89 4.87E−06 0.001002 ENSG00000156298 TSPAN7 −1.50 3.22 20.81 5.08E−06 0.001031 ENSG00000037280 FLT4 −0.95 6.22 20.66 5.48E−06 0.001066 ENSG00000118515 SGK1 1.02 6.19 20.70 5.37E−06 0.001066 ENSG00000171105 INSR −0.82 5.20 20.67 5.47E−06 0.001066 ENSG00000128917 DLL4 −1.06 6.99 20.55 5.81E−06 0.001116 ENSG00000164284 GRPEL2 0.71 5.83 20.28 6.70E−06 0.001268 ENSG00000130449 ZSWIM6 0.84 6.22 20.20 6.96E−06 0.0013 ENSG00000165029 ABCA1 −1.20 4.86 20.18 7.06E−06 0.001303 ENSG00000143127 ITGA10 −0.95 4.84 20.08 7.43E−06 0.001336 ENSG00000187720 THSD4 0.77 5.68 20.08 7.43E−06 0.001336 ENSG00000073008 PVR 0.69 7.34 19.90 8.14E−06 0.001422 ENSG00000107731 UNC5B −1.58 3.90 19.93 8.03E−06 0.001422 ENSG00000122786 CALD1 0.71 9.31 19.89 8.21E−06 0.001422 ENSG00000110092 CCND1 0.77 8.26 19.76 8.76E−06 0.0015 ENSG00000154734 ADAMTS1 −1.32 3.65 19.66 9.23E−06 0.001561 ENSG00000142178 SIK1 1.16 2.11 19.62 9.46E−06 0.001581 ENSG00000168843 FSTL5 1.55 0.35 19.53 9.92E−06 0.001638 ENSG00000121858 TNFSF10 −0.94 4.92 19.41 1.05E−05 0.001722 ENSG00000169213 RAB3B 0.71 5.47 19.35 1.09E−05 0.001754 ENSG00000166670 MMP10 1.61 6.00 19.29 1.12E−05 0.00179 ENSG00000138347 MYPN 2.35 −1.12 19.03 1.29E−05 0.002029 ENSG00000145247 OCIAD2 0.76 5.62 18.90 1.38E−05 0.002126 ENSG00000160801 PTH1R −2.70 −1.67 18.89 1.38E−05 0.002126 ENSG00000184113 CLDN5 −0.91 7.73 18.88 1.39E−05 0.002126 ENSG00000152518 ZFP36L2 −0.62 6.87 18.76 1.48E−05 0.002235 ENSG00000171617 ENC1 0.87 6.92 18.56 1.64E−05 0.002457 ENSG00000166833 NAV2 0.61 5.37 18.42 1.77E−05 0.00262 ENSG00000177076 ACER2 −1.05 3.11 18.28 1.91E−05 0.002793 ENSG00000154928 EPHB1 1.07 2.28 18.16 2.03E−05 0.002915 ENSG00000162407 PLPP3 −0.99 5.19 18.16 2.03E−05 0.002915 ENSG00000099204 ABLIM1 −0.73 8.31 18.11 2.09E−05 0.002957 ENSG00000177606 JUN 0.68 7.59 18.09 2.10E−05 0.002957 ENSG00000170006 TMEM154 0.74 5.48 18.03 2.17E−05 0.003026 ENSG00000118762 PKD2 −0.61 5.46 17.91 2.32E−05 0.003197 ENSG00000108387 ‘SEPT4 −1.64 −0.22 17.45 2.96E−05 0.004038 ENSG00000170891 CYTL1 0.91 3.83 17.25 3.28E−05 0.004439 ENSG00000175040 CHST2 0.88 4.58 17.17 3.42E−05 0.004583 ENSG00000167037 SGSM1 −1.41 1.33 17.15 3.45E−05 0.004587 ENSG00000185070 FLRT2 0.70 7.60 17.09 3.57E−05 0.004694 ENSG00000180304 OAZ2 −0.56 6.82 16.78 4.20E−05 0.005473 ENSG00000257093 KIAA1147 −0.59 7.24 16.71 4.36E−05 0.005628 ENSG00000184897 H1FX −0.58 6.28 16.64 4.53E−05 0.005794 ENSG00000075426 FOSL2 0.57 6.66 16.52 4.80E−05 0.006094 ENSG00000105825 TFPI2 0.57 7.14 16.40 5.13E−05 0.006449 ENSG00000135318 NT5E 0.61 7.62 16.38 5.18E−05 0.00645 ENSG00000127533 F2RL3 1.23 1.06 16.34 5.30E−05 0.006492 ENSG00000205683 DPF3 1.21 2.98 16.34 5.30E−05 0.006492 ENSG00000105499 PLA2G4C 0.76 3.68 16.29 5.44E−05 0.006599 ENSG00000167984 NLRC3 0.99 2.82 16.02 6.26E−05 0.007507 ENSG00000168685 IL7R 1.30 1.79 16.00 6.32E−05 0.007507 ENSG00000172985 SH3RF3 0.64 5.05 16.00 6.34E−05 0.007507 ENSG00000108551 RASD1 1.21 2.55 15.95 6.49E−05 0.00762 ENSG00000081087 OSTM1 0.67 5.66 15.81 6.99E−05 0.008139 ENSG00000128849 CGNL1 −0.78 6.46 15.61 7.78E−05 0.008985 ENSG00000133816 MICAL2 0.69 7.58 15.59 7.87E−05 0.009013 ENSG00000132965 ALOX5AP 2.25 −1.61 15.55 8.04E−05 0.009132 ENSG00000084734 GCKR −2.84 −1.71 15.36 8.89E−05 0.009794 ENSG00000108854 SMURF2 0.53 7.94 15.38 8.81E−05 0.009794 ENSG00000111859 NEDD9 0.52 6.27 15.36 8.89E−05 0.009794 ENSG00000154678 PDE1C 0.89 3.99 15.37 8.84E−05 0.009794 ENSG00000033867 SLC4A7 0.58 7.03 15.33 9.05E−05 0.009889 ENSG00000152207 CYSLTR2 −2.45 −0.56 15.25 9.42E−05 0.010156 ENSG00000188452 CERKL 1.26 0.91 15.25 9.44E−05 0.010156 ENSG00000105357 MYH14 1.43 0.19 15.20 9.66E−05 0.010323 ENSG00000185432 METTL7A −1.01 3.66 15.17 9.83E−05 0.010418 ENSG00000004799 PDK4 −1.18 2.61 15.12 0.000101 0.010639 ENSG00000122694 GLIPR2 0.65 4.88 15.10 0.000102 0.010655 ENSG00000213402 PTPRCAP −3.20 −2.40 15.08 0.000103 0.010665 ENSG00000115902 SLC1A4 −0.69 4.85 14.96 0.00011 0.011119 ENSG00000169248 CXCL11 1.35 0.90 14.97 0.000109 0.011119 ENSG00000189367 KIAA0408 1.83 −0.79 14.96 0.00011 0.011119 ENSG00000102755 FLT1 −0.77 8.53 14.90 0.000114 0.011449 ENSG00000135905 DOCK10 0.62 5.30 14.86 0.000116 0.01158 ENSG00000079102 RUNX1T1 −0.82 4.50 14.83 0.000118 0.011588 ENSG00000153208 MERTK 0.58 5.53 14.83 0.000118 0.011588 ENSG00000198435 NRARP −0.63 5.05 14.82 0.000118 0.011588 ENSG00000141682 PMAIP1 1.02 4.25 14.80 0.00012 0.01165 ENSG00000011347 SYT7 −1.86 −0.09 14.71 0.000126 0.012144 ENSG00000135842 FAM129A −1.24 1.66 14.52 0.000139 0.013339 ENSG00000020577 SAMD4A 0.62 5.94 14.47 0.000143 0.013504 ENSG00000143153 ATP1B1 0.74 5.54 14.47 0.000142 0.013504 ENSG00000177666 PNPLA2 −0.55 6.36 14.43 0.000145 0.013671 ENSG00000085276 MECOM −0.53 7.33 14.39 0.000148 0.013802 ENSG00000111145 ELK3 −0.64 8.64 14.39 0.000149 0.013802 ENSG00000140675 SLC5A2 −5.88 −3.18 14.34 0.000152 0.014056 ENSG00000005108 THSD7A −0.61 5.99 14.27 0.000159 0.014543 ENSG00000157613 CREB3L1 1.04 1.76 14.23 0.000162 0.014743 ENSG00000163584 RPL22L1 −0.61 5.30 14.18 0.000166 0.015011 ENSG00000169418 NPR1 −1.45 3.01 14.12 0.000171 0.015403 ENSG00000131016 AKAP12 0.59 10.29 14.07 0.000176 0.015751 ENSG00000141298 SSH2 0.55 6.75 13.98 0.000184 0.016299 ENSG00000163131 CTSS 0.96 2.75 13.98 0.000185 0.016299 ENSG00000026508 CD44 0.69 5.54 13.94 0.000189 0.016335 ENSG00000136404 TM6SF1 0.69 4.25 13.92 0.00019 0.016335 ENSG00000144583 ‘MARCH4 0.82 5.53 13.95 0.000188 0.016335 ENSG00000175899 A2M −1.56 2.41 13.92 0.000191 0.016335 ENSG00000176749 CDK5R1 1.01 2.89 13.94 0.000189 0.016335 ENSG00000258984 UBE2F-SCLY 3.28 −1.94 13.88 0.000194 0.016538 ENSG00000136960 ENPP2 −1.14 1.35 13.86 0.000197 0.016639 ENSG00000140416 TPM1 0.61 8.65 13.81 0.000202 0.016977 ENSG00000112541 PDE10A −0.92 3.09 13.76 0.000208 0.017288 ENSG00000198286 CARD11 0.59 5.90 13.76 0.000208 0.017288 ENSG00000108825 PTGES3L-AARSD1 1.90 −0.91 13.67 0.000218 0.017886 ENSG00000185650 ZFP36L1 0.51 6.82 13.66 0.000219 0.017886 ENSG00000188290 HES4 −1.30 2.20 13.66 0.000219 0.017886 ENSG00000114631 PODXL2 0.66 4.05 13.62 0.000224 0.018189 ENSG00000106852 LHX6 0.94 4.20 13.59 0.000227 0.018332 ENSG00000171115 GIMAP8 −0.63 7.66 13.56 0.000231 0.018514 ENSG00000189120 SP6 1.04 1.77 13.54 0.000234 0.018568 ENSG00000265972 TXNIP −1.35 3.74 13.54 0.000234 0.018568 ENSG00000154639 CXADR 0.57 4.98 13.51 0.000237 0.018606 ENSG00000169242 EFNA1 −0.75 6.23 13.52 0.000237 0.018606 ENSG00000204304 PBX2 −0.72 5.80 13.49 0.00024 0.018755 ENSG00000157557 ETS2 0.56 6.59 13.45 0.000244 0.018975 ENSG00000105855 ITGB8 1.01 3.67 13.44 0.000246 0.018999 ENSG00000144802 NFKBIZ 0.88 4.33 13.37 0.000255 0.019615 ENSG00000204262 COL5A2 0.51 8.22 13.33 0.000262 0.019978 ENSG00000163092 XIRP2 2.56 −0.54 13.31 0.000264 0.020085 ENSG00000049540 ELN −2.37 −1.72 13.23 0.000275 0.020704 ENSG00000166341 DCHS1 0.50 7.06 13.24 0.000274 0.020704 ENSG00000115414 FN1 0.69 12.30 13.17 0.000284 0.021259 ENSG00000094880 CDC23 −0.63 5.49 13.10 0.000295 0.021903 ENSG00000164647 STEAP1 −0.67 4.33 13.10 0.000296 0.021903 ENSG00000119681 LTBP2 0.48 9.54 13.03 0.000306 0.022511 ENSG00000107562 CXCL12 2.40 −1.46 12.98 0.000314 0.022927 ENSG00000138448 ITGAV 0.55 8.76 12.96 0.000318 0.022927 ENSG00000160223 ICOSLG −0.72 3.61 12.95 0.00032 0.022927 ENSG00000163644 PPM1K −0.64 3.93 12.96 0.000318 0.022927 ENSG00000186575 NF2 0.58 6.53 12.95 0.000319 0.022927 ENSG00000109906 ZBTB16 −1.08 1.12 12.94 0.000322 0.022986 ENSG00000144476 ACKR3 −0.77 4.06 12.89 0.00033 0.023468 ENSG00000136011 STAB2 −3.99 −2.80 12.86 0.000335 0.023698 ENSG00000074181 NOTCH3 −1.01 1.47 12.84 0.000338 0.023798 ENSG00000168916 ZNF608 −0.63 5.15 12.72 0.000362 0.025298 ENSG00000076351 SLC46A1 0.82 2.77 12.71 0.000364 0.025315 ENSG00000151474 FRMD4A 0.53 7.31 12.66 0.000374 0.025951 ENSG00000139508 SLC46A3 −1.45 4.08 12.58 0.00039 0.026876 ENSG00000131386 GALNT15 −1.70 0.16 12.53 0.000401 0.027499 ENSG00000101017 CD40 −0.65 4.42 12.49 0.000409 0.027854 ENSG00000270316 BORCS7-ASMT −6.14 −3.00 12.49 0.00041 0.027854 ENSG00000141668 CBLN2 2.75 0.19 12.48 0.000412 0.027879 ENSG00000146072 TNFRSF21 0.47 6.64 12.44 0.000419 0.028234 ENSG00000183287 CCBE1 1.06 1.39 12.39 0.000432 0.028931 ENSG00000029534 ANK1 −1.37 0.01 12.37 0.000436 0.029068 ENSG00000124593 AL365205.1 −0.90 3.18 12.35 0.00044 0.029134 ENSG00000281028 AC104662.2 −6.09 −3.04 12.35 0.000441 0.029134 ENSG00000187942 LDLRAD2 −0.76 3.41 12.33 0.000446 0.029332 ENSG00000071246 VASH1 −0.66 7.76 12.30 0.000453 0.029655 ENSG00000269307 AC010463.1 −6.06 −3.06 12.27 0.000461 0.030027 ENSG00000050405 LIMA1 0.47 7.26 12.26 0.000463 0.030037 ENSG00000259171 AL163636.2 −1.37 1.58 12.25 0.000465 0.030049 ENSG00000048740 CELF2 0.49 6.23 12.18 0.000483 0.030936 ENSG00000116774 OLFML3 0.99 1.45 12.18 0.000482 0.030936 ENSG00000182240 BACE2 −0.56 7.52 12.17 0.000486 0.030971 ENSG00000204767 FAM196B 0.77 3.39 12.15 0.00049 0.031102 ENSG00000211448 DIO2 2.26 −1.84 12.14 0.000494 0.031198 ENSG00000151468 CCDC3 −1.17 0.72 12.11 0.000501 0.031481 ENSG00000196498 NCOR2 0.52 7.49 12.09 0.000506 0.031651 ENSG00000049130 KITLG 0.70 5.16 12.07 0.000512 0.031901 ENSG00000137033 IL33 −1.94 4.40 12.06 0.000516 0.031997 ENSG00000130635 COL5A1 0.52 8.58 12.03 0.000524 0.032215 ENSG00000133056 PIK3C2B −0.67 5.90 12.03 0.000522 0.032215 ENSG00000116741 RGS2 0.79 3.02 12.02 0.000528 0.0323 ENSG00000129757 CDKN1C −0.95 2.05 11.98 0.000536 0.032564 ENSG00000179195 ZNF664 −0.66 6.99 11.99 0.000535 0.032564 ENSG00000185924 RTN4RL1 −2.85 −2.54 11.97 0.000542 0.032704 ENSG00000189060 H1F0 −0.50 6.44 11.96 0.000543 0.032704 ENSG00000255767 AC108488.2 5.64 −3.35 11.95 0.000548 0.032834 ENSG00000108691 CCL2 0.56 5.05 11.89 0.000566 0.033765 ENSG00000106069 CHN2 0.83 2.52 11.87 0.000571 0.033914 ENSG00000132702 HAPLN2 1.07 3.28 11.85 0.000575 0.033914 ENSG00000183691 NOG 1.96 0.87 11.85 0.000575 0.033914 ENSG00000105738 SIPA1L3 0.53 5.37 11.84 0.000578 0.033924 ENSG00000154096 THY1 −1.73 0.37 11.84 0.00058 0.033924 ENSG00000140937 CDH11 0.58 5.50 11.80 0.000592 0.034495 ENSG00000127241 MASP1 −1.60 −0.50 11.77 0.000601 0.034815 ENSG00000164574 GALNT10 0.47 7.18 11.77 0.000603 0.034815 ENSG00000259112 NDUFC2-KCTD14 5.59 −3.38 11.75 0.000609 0.035044 ENSG00000109046 WSB1 −0.57 8.59 11.72 0.000619 0.035055 ENSG00000130054 FAM155B 1.73 −1.28 11.72 0.000619 0.035055 ENSG00000133401 PDZD2 −0.68 3.72 11.72 0.000618 0.035055 ENSG00000284041 AC073111.3 −5.87 −3.18 11.72 0.000619 0.035055 ENSG00000115008 IL1A 1.56 1.13 11.67 0.000634 0.035722 ENSG00000185737 NRG3 −0.86 2.50 11.66 0.000638 0.035834 ENSG00000196923 PDLIM7 0.57 7.15 11.64 0.000644 0.036026 ENSG00000187583 PLEKHN1 1.60 −1.07 11.62 0.000652 0.036337 ENSG00000135324 MRAP2 −0.97 3.21 11.57 0.000672 0.037286 ENSG00000158186 MRAS 0.56 4.27 11.55 0.000678 0.037503 ENSG00000092969 TGFB2 1.40 3.41 11.54 0.000682 0.037523 ENSG00000158373 HIST1H2BD 1.95 0.56 11.53 0.000684 0.037523 ENSG00000118946 PCDH17 1.00 3.06 11.49 0.0007 0.038238 ENSG00000067798 NAV3 0.64 5.24 11.48 0.000705 0.038357 ENSG00000143344 RGL1 0.81 6.48 11.46 0.00071 0.03848 ENSG00000188042 ARL4C 0.65 3.97 11.43 0.000723 0.039053 ENSG00000162772 ATF3 0.98 1.96 11.41 0.000731 0.039331 ENSG00000164104 HMGB2 −0.50 7.51 11.38 0.000742 0.039771 ENSG00000138411 HECW2 0.47 7.31 11.36 0.000749 0.03998 ENSG00000142627 EPHA2 0.49 7.57 11.33 0.000763 0.040464 ENSG00000258947 TUBB3 0.62 7.55 11.33 0.000761 0.040464 ENSG00000183775 KCTD16 1.30 −0.12 11.30 0.000773 0.040837 ENSG00000114948 ADAM23 0.55 5.15 11.26 0.000793 0.041703 ENSG00000114315 HES1 −0.49 5.56 11.22 0.000811 0.042315 ENSG00000138772 ANXA3 0.65 5.85 11.22 0.000808 0.042315 ENSG00000270276 HIST2H4B −1.49 −0.13 11.21 0.000813 0.042315 ENSG00000213694 S1PR3 0.71 4.78 11.16 0.000835 0.043284 ENSG00000150687 PRSS23 0.44 9.70 11.15 0.000841 0.043462 ENSG00000095303 PTGS1 0.62 4.28 11.12 0.000852 0.043854 ENSG00000146674 IGFBP3 −0.93 1.59 11.11 0.00086 0.04411 ENSG00000109436 TBC1D9 0.45 6.66 11.09 0.000868 0.044376 ENSG00000274933 TBC1D3I 3.01 −2.32 11.07 0.000879 0.044738 ENSG00000116678 LEPR −0.62 4.60 11.04 0.000893 0.044947 ENSG00000156642 NPTN −0.65 6.37 11.04 0.000892 0.044947 ENSG00000198720 ANKRD13B 0.53 4.45 11.03 0.000895 0.044947 ENSG00000228144 AC078927.1 5.57 −3.50 11.04 0.000891 0.044947 ENSG00000256966 AL513165.2 2.50 −2.24 11.01 0.000907 0.045379 ENSG00000156920 ADGRG4 3.17 −2.89 10.97 0.000926 0.046138 ENSG00000171877 FRMD5 0.66 4.15 10.92 0.00095 0.047183 ENSG00000164946 FREM1 1.21 0.17 10.87 0.000975 0.048254 ENSG00000159640 ACE −0.99 5.64 10.86 0.000982 0.048417 ENSG00000176771 NCKAP5 1.30 0.36 10.84 0.000993 0.048822 ENSG00000198513 ATL1 1.00 3.22 10.83 0.000999 0.048924 ENSG00000100234 TIMP3 −0.98 3.13 10.82 0.001006 0.049108

Table 4 lists genes differentially expressed in THSD1 knockdown HUVECs

TABLE 4 Differentially Expressed Genes in THSD1 Knockdown HBMECs Ensembl Gene Gene Symbol logFC logCPM LR PValue FDR ENSG00000117152 RGS4 −1.59 6.07 88.46 5.18E−21 7.37E−17 ENSG00000272949 AC093668.2 8.74 −0.80 64.63 9.02E−16 6.40E−12 ENSG00000283088 AC010487.3 −8.71 −0.85 60.76 6.45E−15 3.05E−11 ENSG00000136114 THSD1 −1.51 4.73 58.41 2.13E−14 7.56E−11 ENSG00000146674 IGFBP3 −2.59 2.50 54.84 1.31E−13 3.72E−10 ENSG00000284057 AP001273.2 8.19 −1.30 52.15 5.15E−13 1.22E−09 ENSG00000133101 CCNA1 −1.31 4.76 45.04 1.93E−11 3.92E−08 ENSG00000078018 MAP2 −1.31 4.98 43.56 4.11E−11 7.29E−08 ENSG00000240583 AQP1 −2.37 3.93 41.78 1.02E−10 1.61E−07 ENSG00000008517 IL32 1.06 6.45 39.73 2.92E−10 4.15E−07 ENSG00000154734 ADAMTS1 −1.55 3.82 38.76 4.78E−10 6.18E−07 ENSG00000068489 PRR11 −1.00 6.19 37.14 1.10E−09 1.30E−06 ENSG00000118777 ABCG2 −1.75 3.44 36.14 1.84E−09 1.87E−06 ENSG00000168542 COL3A1 2.68 1.64 36.27 1.72E−09 1.87E−06 ENSG00000138180 CEP55 −0.99 6.19 35.71 2.30E−09 2.17E−06 ENSG00000272414 FAM47E-STBD1 4.42 −1.05 35.52 2.52E−09 2.24E−06 ENSG00000264187 AC055811.2 7.52 −1.89 35.39 2.70E−09 2.26E−06 ENSG00000072571 HMMR −1.09 5.70 34.95 3.39E−09 2.67E−06 ENSG00000112984 KIF20A −1.03 6.21 34.59 4.07E−09 3.05E−06 ENSG00000126787 DLGAP5 −0.96 6.86 34.15 5.09E−09 3.62E−06 ENSG00000081087 OSTM1 1.10 5.56 33.06 8.93E−09 6.04E−06 ENSG00000117399 CDC20 −1.02 6.40 32.40 1.25E−08 8.08E−06 ENSG00000100292 HMOX1 0.94 6.83 31.53 1.96E−08 1.21E−05 ENSG00000134057 CCNB1 −0.94 6.95 31.40 2.10E−08 1.24E−05 ENSG00000143228 NUF2 −1.06 5.01 30.90 2.71E−08 1.48E−05 ENSG00000166851 PLK1 −1.03 6.17 30.91 2.70E−08 1.48E−05 ENSG00000108691 CCL2 1.38 5.33 29.45 5.74E−08 2.86E−05 ENSG00000132470 ITGB4 −1.94 3.69 29.41 5.84E−08 2.86E−05 ENSG00000164104 HMGB2 −0.86 7.35 29.54 5.47E−08 2.86E−05 ENSG00000163661 PTX3 −1.01 7.71 29.11 6.85E−08 3.24E−05 ENSG00000145386 CCNA2 −1.01 6.45 29.03 7.12E−08 3.26E−05 ENSG00000142945 KIF2C −0.95 5.36 28.79 8.07E−08 3.58E−05 ENSG00000146678 IGFBP1 −2.51 0.45 28.44 9.65E−08 4.15E−05 ENSG00000137812 KNL1 −1.09 5.81 28.26 1.06E−07 4.44E−05 ENSG00000138182 KIF20B −1.01 5.81 28.10 1.15E−07 4.67E−05 ENSG00000080986 NDC80 −1.05 5.15 27.56 1.52E−07 5.90E−05 ENSG00000136928 GABBR2 −0.95 6.03 27.44 1.62E−07 5.90E−05 ENSG00000137804 NUSAP1 −0.95 6.08 27.46 1.60E−07 5.90E−05 ENSG00000273294 C1QTNF3-AMACR 7.05 −2.28 27.45 1.62E−07 5.90E−05 ENSG00000131747 TOP2A −0.97 8.09 26.97 2.07E−07 7.35E−05 ENSG00000087586 AURKA −0.86 5.67 26.90 2.14E−07 7.42E−05 ENSG00000094880 CDC23 −0.93 5.49 26.82 2.24E−07 7.43E−05 ENSG00000161888 SPC24 −1.08 4.35 26.81 2.25E−07 7.43E−05 ENSG00000066279 ASPM −1.18 6.89 26.71 2.36E−07 7.62E−05 ENSG00000198901 PRC1 −0.91 6.85 26.11 3.23E−07 0.000102 ENSG00000088325 TPX2 −0.86 7.28 25.96 3.49E−07 0.000108 ENSG00000138778 CENPE −1.03 6.40 25.44 4.58E−07 0.000136 ENSG00000140525 FANCI −0.85 5.87 25.43 4.60E−07 0.000136 ENSG00000111206 FOXM1 −0.80 6.61 25.29 4.94E−07 0.000143 ENSG00000076382 SPAG5 −0.83 5.81 25.10 5.46E−07 0.000155 ENSG00000170312 CDK1 −0.95 6.03 25.05 5.59E−07 0.000156 ENSG00000161800 RACGAP1 −0.88 5.47 24.90 6.04E−07 0.000162 ENSG00000168078 PBK −0.93 5.37 24.91 6.02E−07 0.000162 ENSG00000146072 TNFRSF21 0.88 6.42 24.77 6.45E−07 0.00017 ENSG00000134690 CDCA8 −1.05 5.25 24.59 7.11E−07 0.000184 ENSG00000148773 MKI67 −1.17 7.99 24.06 9.33E−07 0.000237 ENSG00000102575 ACP5 −3.55 2.81 24.03 9.50E−07 0.000237 ENSG00000024526 DEPDC1 −0.99 5.61 23.90 1.02E−06 0.000249 ENSG00000099937 SERPIND1 −1.85 2.87 23.80 1.07E−06 0.000258 ENSG00000276612 FP565260.2 −6.67 −2.61 23.74 1.10E−06 0.000261 ENSG00000118193 KIF14 −1.04 5.04 23.64 1.16E−06 0.000266 ENSG00000175063 UBE2C −1.03 5.18 23.65 1.16E−06 0.000266 ENSG00000123485 HJURP −0.92 4.98 23.46 1.28E−06 0.000286 ENSG00000157456 CCNB2 −0.84 5.61 23.41 1.31E−06 0.000286 ENSG00000163584 RPL22L1 −0.90 5.95 23.41 1.31E−06 0.000286 ENSG00000117724 CENPF −0.96 7.44 22.28 2.35E−06 0.000506 ENSG00000100297 MCM5 −0.86 6.30 22.12 2.56E−06 0.000535 ENSG00000105357 MYH14 4.21 −0.61 22.15 2.53E−06 0.000535 ENSG00000138160 KIF11 −0.95 6.34 22.01 2.71E−06 0.000558 ENSG00000093009 CDC45 −1.00 4.55 21.97 2.77E−06 0.000562 ENSG00000185070 FLRT2 0.76 8.29 21.87 2.92E−06 0.000584 ENSG00000116774 OLFML3 1.52 2.30 21.78 3.06E−06 0.000603 ENSG00000123689 G0S2 −1.97 2.29 21.72 3.15E−06 0.000613 ENSG00000167900 TK1 −0.76 5.68 21.56 3.43E−06 0.000655 ENSG00000184661 CDCA2 −0.93 4.87 21.54 3.46E−06 0.000655 ENSG00000013810 TACC3 −0.76 6.36 21.52 3.50E−06 0.000655 ENSG00000101057 MYBL2 −0.78 6.00 21.42 3.70E−06 0.000682 ENSG00000112742 TTK −1.03 5.14 21.29 3.95E−06 0.000719 ENSG00000163808 KIF15 −1.04 4.74 21.00 4.59E−06 0.000826 ENSG00000011426 ANLN −1.01 7.23 20.77 5.19E−06 0.00091 ENSG00000265107 GJA5 −6.45 −2.77 20.77 5.19E−06 0.00091 ENSG00000121152 NCAPH −0.96 4.79 20.73 5.28E−06 0.000915 ENSG00000114631 PODXL2 1.26 3.20 20.67 5.44E−06 0.000932 ENSG00000129173 E2F8 −1.04 4.57 20.50 5.97E−06 0.00101 ENSG00000101335 MYL9 0.82 5.36 20.46 6.10E−06 0.00102 ENSG00000164109 MAD2L1 −0.92 5.54 20.32 6.55E−06 0.001082 ENSG00000104738 MCM4 −0.92 6.63 20.27 6.74E−06 0.0011 ENSG00000169679 BUB1 −0.99 5.93 20.11 7.32E−06 0.001182 ENSG00000073111 MCM2 −0.82 5.76 19.94 7.98E−06 0.00126 ENSG00000165480 SKA3 −0.99 4.57 19.95 7.95E−06 0.00126 ENSG00000075218 GTSE1 −0.89 5.40 19.81 8.54E−06 0.001333 ENSG00000267618 AC004223.3 6.30 −2.86 19.78 8.70E−06 0.001344 ENSG00000163131 CTSS 1.44 3.29 19.60 9.54E−06 0.001458 ENSG00000089685 BIRC5 −0.77 6.29 19.52 9.98E−06 0.001508 ENSG00000178999 AURKB −0.98 4.85 19.36 1.08E−05 0.001618 ENSG00000186193 SAPCD2 −0.87 4.80 19.32 1.10E−05 0.001632 ENSG00000071539 TRIP13 −0.84 5.48 19.22 1.17E−05 0.001707 ENSG00000156504 FAM122B −0.84 4.71 19.18 1.19E−05 0.001722 ENSG00000165092 ALDH1A1 0.69 6.16 19.14 1.21E−05 0.001741 ENSG00000123975 CKS2 −0.80 5.71 19.02 1.29E−05 0.001764 ENSG00000135476 ESPL1 −0.97 4.76 19.09 1.25E−05 0.001764 ENSG00000173597 SULT1B1 −0.97 7.01 19.02 1.29E−05 0.001764 ENSG00000189431 RASSF10 −3.28 −1.94 19.03 1.29E−05 0.001764 ENSG00000237649 KIFC1 −0.93 5.31 19.05 1.27E−05 0.001764 ENSG00000122966 CIT −0.81 5.46 18.93 1.36E−05 0.001836 ENSG00000117650 NEK2 −0.85 4.48 18.90 1.38E−05 0.001846 ENSG00000100526 CDKN3 −0.93 4.10 18.77 1.47E−05 0.001954 ENSG00000156970 BUB1B −0.93 5.84 18.68 1.55E−05 0.002039 ENSG00000284041 AC073111.3 −6.19 −2.96 18.66 1.57E−05 0.002041 ENSG00000115163 CENPA −1.06 3.87 18.25 1.94E−05 0.002507 ENSG00000269891 ARHGAP19-SLIT1 6.14 −2.97 18.05 2.15E−05 0.002751 ENSG00000065328 MCM10 −1.01 4.18 17.99 2.22E−05 0.002822 ENSG00000125378 BMP4 0.74 6.11 17.97 2.25E−05 0.002823 ENSG00000079616 KIF22 −0.75 5.65 17.89 2.35E−05 0.002915 ENSG00000151640 DPYSL4 0.98 4.09 17.87 2.36E−05 0.002915 ENSG00000053747 LAMA3 −0.87 4.23 17.67 2.62E−05 0.003214 ENSG00000154175 ABI3BP −0.84 5.18 17.65 2.65E−05 0.003219 ENSG00000183856 IQGAP3 −0.81 5.10 17.63 2.68E−05 0.003226 ENSG00000198826 ARHGAP11A −0.92 6.08 17.48 2.91E−05 0.003475 ENSG00000228716 DHFR −0.74 6.16 17.44 2.97E−05 0.003512 ENSG00000076003 MCM6 −0.84 6.10 17.39 3.05E−05 0.003576 ENSG00000173166 RAPH1 −0.94 5.58 17.28 3.22E−05 0.003754 ENSG00000123473 STIL −0.90 4.92 17.23 3.31E−05 0.003819 ENSG00000117595 IRF6 1.52 3.34 17.10 3.55E−05 0.004062 ENSG00000146918 NCAPG2 −0.77 5.94 17.06 3.63E−05 0.004089 ENSG00000164647 STEAP1 −0.83 5.07 17.07 3.61E−05 0.004089 ENSG00000196878 LAMB3 −0.85 4.47 17.03 3.67E−05 0.004108 ENSG00000171241 SHCBP1 −0.86 5.58 17.01 3.72E−05 0.004126 ENSG00000150630 VEGFC −0.96 4.78 16.89 3.95E−05 0.004353 ENSG00000090889 KIF4A −0.77 5.75 16.62 4.56E−05 0.004974 ENSG00000121621 KIF18A −0.92 4.27 16.61 4.59E−05 0.004974 ENSG00000101447 FAM83D −0.88 5.40 16.54 4.77E−05 0.005136 ENSG00000169604 ANTXR1 1.03 4.62 16.47 4.93E−05 0.005267 ENSG00000096060 FKBP5 −0.68 6.90 16.31 5.38E−05 0.005672 ENSG00000167434 CA4 −4.19 −2.69 16.31 5.39E−05 0.005672 ENSG00000163092 XIRP2 1.93 1.44 16.29 5.43E−05 0.005675 ENSG00000104147 OIP5 −1.14 2.91 16.25 5.56E−05 0.005762 ENSG00000119403 PHF19 −0.64 6.01 16.19 5.74E−05 0.005868 ENSG00000211448 DIO2 1.64 0.55 16.20 5.71E−05 0.005868 ENSG00000185432 METTL7A −0.90 5.20 16.11 5.99E−05 0.006078 ENSG00000105499 PLA2G4C 1.03 3.47 16.09 6.05E−05 0.006091 ENSG00000139734 DIAPH3 −0.80 5.47 16.01 6.30E−05 0.0063 ENSG00000163453 IGFBP7 0.68 7.65 15.92 6.60E−05 0.006561 ENSG00000140416 TPM1 0.64 8.27 15.87 6.77E−05 0.006677 ENSG00000109805 NCAPG −0.77 6.26 15.73 7.30E−05 0.007153 ENSG00000137807 KIF23 −0.78 5.95 15.66 7.59E−05 0.007335 ENSG00000179195 ZNF664 −0.88 6.90 15.66 7.58E−05 0.007335 ENSG00000144554 FANCD2 −0.87 4.59 15.57 7.95E−05 0.007632 ENSG00000101003 GINS1 −0.83 4.79 15.51 8.19E−05 0.007811 ENSG00000137310 TCF19 −0.81 4.92 15.35 8.92E−05 0.008445 ENSG00000115008 IL1A 1.97 0.90 15.32 9.06E−05 0.008525 ENSG00000163751 CPA3 1.34 1.70 15.30 9.18E−05 0.008576 ENSG00000197632 SERPINB2 −1.60 3.06 15.21 9.61E−05 0.008924 ENSG00000133119 RFC3 −0.80 4.52 15.16 9.87E−05 0.009103 ENSG00000140545 MFGE8 0.73 5.90 15.12 0.00010086 0.009244 ENSG00000101188 NTSR1 −1.16 2.79 15.08 0.00010312 0.009391 ENSG00000171848 RRM2 −1.18 6.87 15.06 0.00010394 0.009404 ENSG00000185480 PARPBP −0.79 4.44 15.05 0.00010467 0.009411 ENSG00000140675 SLC5A2 4.89 −2.99 14.82 0.00011808 0.010484 ENSG00000151725 CENPU −0.74 4.67 14.83 0.00011777 0.010484 ENSG00000168243 GNG4 2.60 −1.74 14.78 0.00012062 0.010643 ENSG00000173207 CKS1B −0.73 5.38 14.77 0.0001214 0.010646 ENSG00000010292 NCAPD2 −0.62 7.04 14.64 0.00012988 0.011319 ENSG00000173281 PPP1R3B 0.64 6.98 14.61 0.0001319 0.011425 ENSG00000164611 PTTG1 −0.64 5.60 14.58 0.00013445 0.011569 ENSG00000167261 DPEP2 2.79 −2.01 14.57 0.00013518 0.011569 ENSG00000184445 KNTC1 −0.70 5.37 14.55 0.00013641 0.011604 ENSG00000012048 BRCA1 −0.90 4.77 14.49 0.00014056 0.011791 ENSG00000092853 CLSPN −0.87 4.95 14.49 0.0001411 0.011791 ENSG00000203668 CHML −0.70 5.13 14.49 0.00014087 0.011791 ENSG00000101868 POLA1 −0.84 4.68 14.45 0.00014375 0.011893 ENSG00000122694 GLIPR2 0.70 5.12 14.45 0.000144 0.011893 ENSG00000149591 TAGLN 1.24 1.91 14.37 0.00015051 0.012359 ENSG00000280537 AC068946.1 2.14 −1.15 14.35 0.00015184 0.012397 ENSG00000105011 ASF1B −0.91 4.72 14.28 0.00015721 0.012762 ENSG00000105889 STEAP1B −0.83 4.64 14.20 0.00016442 0.013271 ENSG00000124721 DNAH8 1.61 3.82 14.17 0.00016717 0.013341 ENSG00000130816 DNMT1 −0.60 7.04 14.17 0.00016671 0.013341 ENSG00000162645 GBP2 0.60 6.15 14.14 0.00016943 0.013447 ENSG00000058804 NDC1 −0.71 5.97 14.03 0.00018026 0.014227 ENSG00000145604 SKP2 −0.67 5.33 14.00 0.0001825 0.014324 ENSG00000123219 CENPK −0.71 4.81 13.98 0.00018513 0.014341 ENSG00000136824 SMC2 −0.81 6.08 13.97 0.00018575 0.014341 ENSG00000188517 COL25A1 2.38 −1.61 13.97 0.00018564 0.014341 ENSG00000107984 DKK1 −0.63 5.85 13.95 0.00018762 0.01437 ENSG00000204262 COL5A2 0.61 8.73 13.95 0.00018814 0.01437 ENSG00000162063 CCNF −0.67 5.31 13.89 0.00019352 0.014701 ENSG00000258064 AC073612.1 −6.56 −2.69 13.76 0.00020816 0.01573 ENSG00000120802 TMPO −0.66 7.44 13.64 0.00022161 0.016569 ENSG00000163507 CIP2A −0.82 5.14 13.64 0.0002212 0.016569 ENSG00000186185 KIF18B −0.86 4.35 13.61 0.00022501 0.016735 ENSG00000149503 INCENP −0.86 5.22 13.41 0.00025051 0.018535 ENSG00000167601 AXL −0.55 7.33 13.28 0.00026806 0.019731 ENSG00000139618 BRCA2 −0.91 4.49 13.19 0.00028185 0.020639 ENSG00000163554 SPTA1 2.18 −0.67 13.16 0.00028663 0.020881 ENSG00000100311 PDGFB 0.82 6.70 13.08 0.00029782 0.021586 ENSG00000106069 CHN2 0.94 3.27 12.98 0.00031547 0.022542 ENSG00000239389 PCDHA13 6.39 −2.80 12.99 0.00031302 0.022542 ENSG00000258947 TUBB3 0.59 7.10 12.97 0.00031577 0.022542 ENSG00000144354 CDCA7 −0.74 4.99 12.94 0.0003212 0.022815 ENSG00000123080 CDKN2C −0.81 4.00 12.93 0.00032414 0.022909 ENSG00000103489 XYLT1 1.13 2.69 12.83 0.00034094 0.023859 ENSG00000178538 CA8 −1.71 0.71 12.83 0.00034088 0.023859 ENSG00000163535 SGO2 −0.80 5.30 12.81 0.00034481 0.024012 ENSG00000133110 POSTN −1.11 3.05 12.78 0.00035075 0.024306 ENSG00000035499 DEPDC1B −0.85 4.24 12.75 0.00035616 0.024325 ENSG00000150540 HNMT 0.80 4.05 12.75 0.0003557 0.024325 ENSG00000176890 TYMS −0.61 6.99 12.75 0.00035522 0.024325 ENSG00000261459 AC002310.5 −6.20 −2.95 12.71 0.00036311 0.024681 ENSG00000117593 DARS2 −0.67 5.54 12.68 0.00036955 0.024999 ENSG00000111341 MGP −0.57 6.67 12.65 0.00037599 0.025314 ENSG00000058085 LAMC2 0.75 4.73 12.57 0.00039222 0.026282 ENSG00000103257 SLC7A5 −0.71 4.74 12.49 0.00040895 0.027275 ENSG00000214357 NEURL1B −1.41 0.86 12.46 0.0004151 0.027556 ENSG00000258555 SPECC1L-ADORA2A 6.16 −2.96 12.37 0.00043612 0.028683 ENSG00000268643 AC006486.1 −6.10 −3.02 12.37 0.00043584 0.028683 ENSG00000186871 ERCC6L −0.97 3.80 12.31 0.00045156 0.029562 ENSG00000128944 KNSTRN −0.64 5.27 12.26 0.00046273 0.029611 ENSG00000134222 PSRC1 −0.89 3.87 12.28 0.00045803 0.029611 ENSG00000142731 PLK4 −0.89 4.91 12.27 0.00046122 0.029611 ENSG00000147536 GINS4 −0.85 4.14 12.28 0.00045741 0.029611 ENSG00000267022 AC067968.1 −2.73 −1.12 12.26 0.00046177 0.029611 ENSG00000155093 PTPRN2 1.40 0.73 12.23 0.00047029 0.029846 ENSG00000188229 TUBB4B −0.69 8.39 12.23 0.00047061 0.029846 ENSG00000215252 GOLGA8B 0.68 4.46 12.21 0.00047536 0.030013 ENSG00000149573 MPZL2 −0.67 5.40 12.20 0.00047877 0.030095 ENSG00000129195 PIMREG −0.79 4.07 12.18 0.00048388 0.030282 ENSG00000152104 PTPN14 −0.60 7.17 12.16 0.00048777 0.030391 ENSG00000197457 STMN3 2.81 3.31 12.14 0.00049373 0.030629 ENSG00000168874 ATOH8 0.84 3.70 12.13 0.00049593 0.030631 ENSG00000255073 ZFP91-CNTF −5.43 −3.45 12.10 0.00050547 0.031085 ENSG00000188486 H2AFX −0.63 6.27 12.02 0.00052646 0.032209 ENSG00000213297 ZNF625-ZNF20 1.41 0.93 12.01 0.00052828 0.032209 ENSG00000102007 PLP2 −0.55 6.27 11.97 0.00053967 0.032496 ENSG00000135842 FAM129A −1.02 3.29 11.97 0.00053985 0.032496 ENSG00000168843 FSTL5 1.09 3.04 11.98 0.00053834 0.032496 ENSG00000178878 APOLD1 −0.89 3.58 11.94 0.00055082 0.033017 ENSG00000113368 LMNB1 −0.66 6.70 11.92 0.00055512 0.033135 ENSG00000137473 TTC29 1.72 −0.20 11.88 0.0005664 0.033666 ENSG00000148848 ADAM12 1.25 1.79 11.86 0.00057272 0.0339 ENSG00000114346 ECT2 −0.68 5.97 11.84 0.00058063 0.034226 ENSG00000196739 COL27A1 0.57 5.97 11.77 0.00060245 0.03522 ENSG00000258677 AC022826.2 −1.79 −0.91 11.77 0.00060175 0.03522 ENSG00000129810 SGO1 −0.89 3.52 11.75 0.00060846 0.035425 ENSG00000198830 HMGN2 −0.53 8.42 11.73 0.00061337 0.035566 ENSG00000174371 EXO1 −0.93 3.73 11.69 0.00062871 0.036307 ENSG00000127564 PKMYT1 −0.90 3.52 11.66 0.00063706 0.03664 ENSG00000100368 CSF2RB 0.55 5.77 11.64 0.00064394 0.036738 ENSG00000135451 TROAP −0.73 4.10 11.64 0.00064385 0.036738 ENSG00000092470 WDR76 −0.72 4.40 11.60 0.00065967 0.037485 ENSG00000143401 ANP32E −0.56 6.78 11.58 0.00066731 0.037768 ENSG00000257411 AC034102.2 1.93 0.75 11.54 0.0006809 0.038384 ENSG00000146670 CDCA5 −0.72 5.02 11.50 0.00069768 0.039175 ENSG00000167325 RRM1 −0.57 6.89 11.47 0.00070846 0.039623 ENSG00000152253 SPC25 −0.89 3.82 11.46 0.00071221 0.039677 ENSG00000122952 ZWINT −0.71 5.53 11.43 0.00072353 0.04015 ENSG00000121966 CXCR4 1.02 5.44 11.40 0.00073256 0.040493 ENSG00000101224 CDC25B −0.56 6.33 11.38 0.00074161 0.040707 ENSG00000136490 LIMD2 0.60 5.23 11.38 0.00074216 0.040707 ENSG00000127528 KLF2 −0.82 4.46 11.35 0.00075267 0.041125 ENSG00000151388 ADAMTS12 0.85 3.45 11.34 0.00075879 0.041163 ENSG00000154839 SKA1 −0.87 4.20 11.34 0.00075917 0.041163 ENSG00000119969 HELLS −0.65 4.77 11.30 0.00077592 0.041911 ENSG00000108055 SMC3 −0.53 6.68 11.28 0.00078172 0.042065 ENSG00000125695 AC046185.1 5.80 −3.20 11.24 0.00079891 0.042828 ENSG00000146411 SLC2A12 1.26 2.09 11.23 0.00080506 0.042888 ENSG00000159167 STC1 −0.93 4.54 11.23 0.00080607 0.042888 ENSG00000181634 TNFSF15 0.78 5.81 11.19 0.00082212 0.043579 ENSG00000111665 CDCA3 −0.78 3.97 11.16 0.00083518 0.044106 ENSG00000067141 NEO1 −2.49 −2.06 11.10 0.00086297 0.045237 ENSG00000099282 TSPAN15 0.66 6.21 11.10 0.00086289 0.045237 ENSG00000120256 LRP11 0.57 5.86 10.97 0.00092765 0.048365 ENSG00000145990 GFOD1 −0.59 5.47 10.96 0.00093285 0.048365 ENSG00000166250 CLMP −1.02 2.47 10.96 0.00093028 0.048365 ENSG00000072501 SMC1A −0.59 6.94 10.94 0.00093894 0.048504 ENSG00000112378 PERP −0.59 7.18 10.92 0.00095053 0.048925 ENSG00000077152 UBE2T −0.67 4.49 10.90 0.00096365 0.049243 ENSG00000198553 KCNRG 1.57 −0.55 10.90 0.00096042 0.049243 ENSG00000120337 TNFSF18 0.87 4.84 10.87 0.00097964 0.049881

Bioinformatic analyses highlighted a role for THSD1 in regulating endothelial to mesenchymal transition.

Example 3: Endothelial Dysfunction Mediated by THSD1 Through the Biological Process of Endothelial to Mesenchymal Transition (EndMT)

Regulation of Endothelial to Mesenchymal Transition as a Target for Treating IA

Endothelial to mesenchymal transition (EndMT) is a biological process whereby an endothelial cell undergoes a series of molecular events that lead to a change in phenotype toward a mesenchymal cell like phenotype. During this process, endothelial cells adopt a mesenchymal phenotype displaying typical mesenchymal cell morphology and functions, including the promotion of inflammatory response. In addition, endothelial cells lose the expression of endothelial cell-specific proteins and initiate the expression of mesenchymal cell-specific genes and the production.

We applied global transcriptomics approaches to identify genes disrupted in THSD1 knockdown studies and obtained RNAseq data for two THSD1 knock-down endothelial cell lines. Both cell lines support the evidence that THSD1 mediates EndMT function (THSD1 Knock-down induces the activations of EMT—loss of endothelial normal function, but increased inflammatory cell), see FIG. 3 .

Example 4: Reagents for Studying the Function of THSD1

A variety of chimeric nucleic acid molecules encoding a nucleic acid sequence of a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele that (a) encodes a THSD1 protein modified to comprise a replacement of a sequence encoding the THSD1 protein or portion thereof with a heterologous missense THSD1 protein or a portion thereof were engineered. First, two synonymous mutations (DM) were introduced into a WT THSD1 sequence to make an siRNA-resistant form of THSD1.

Next, each individual rare variant associated with pathogenesis was made by PCR mutagenesis and subcloned into pLVX-TetOne vector with a C-terminal tagging of FLAG. pLVX-TetOne vector is a lentiviral vector and can be induced by addition of doxycycline. The constructs created for the experiment included chimeric nucleic acid molecules wherein the heterologous missense THSD1 protein or a portion thereof comprised (i) a single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L5F) substitution; (ii) a single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution; (iii) a single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution; (iv) a single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution; (v) a single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution; (vi) a single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution; (vii) a single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution; (viii) any combination of (i)-(vii).

Table 5 illustrates exemplary nucleic acids constructs made:

TABLE 5 1 pLVX-TetOne-THSD1-DM-2FLAG-WT 2 pLVX-TetOne-THSD1-DM-2FLAG-L5F 3 pLVX-TetOne-THSD1-DM-2FLAG-R460W 4 pLVX-TetOne-THSD1-DM-2FLAG-E466G 5 pLVX-TetOne-THSD1-DM-2FLAG-G600E 6 pLVX-TetOne-THSD1-DM-2FLAG-P639L 7 pLVX-TetOne-THSD1-DM-2FLAG-P639L 8 pLVX-TetOne-THSD1-DM-2FLAG-S775P

Next, the expression of THSD1 rare variants in human brain endothelial cells. A pLVX-TetOne backbone was used to generate lentiviruses together with co-plasmids psPAX2 (2 ug) and pMD2.G (2 ug). Human brain endothelial cells (D3) were infected by lentiviruses and the cells stably expressing each variant were selected against puromycin (8 ug/ml) for 3 days. Each stable cell line was then induced by doxycycline/DOX (10 ng/ml) for 2 days and whole cell lysates were collected for examining protein expression by Western blot against FLAG tag. See FIG. 4 . All of the THSD1 variants were well expressed in D3 cells after DOX induction. These lines can serve as tools to study the functions of THSD1 rare variants in brain endothelial cells, which have been associated with intracranial aneurysm development in humans.

Example 6: Genetic Engineering of Non-Human Animal Models Encoding Distinct THSD1 Rare Variants for In-Vivo Assessment of Therapies

Identified Conserved Sequences Suitable for Assessing Cross Species Efficacies of Treatments:

A protein alignment between human and zebrafish THSD1 was performed. See FIG. 5 illustrates the result of the alignment with black color indicating identical amino acids and the grey color indicating similar amino acids between humans and zebra fish. The alignment underscored that three rare variants of THSD1 identified in the aforementioned examples were conserved between human and zebrafish. The conserved amino acids include R450, R460 and T653. See, e.g., FIG. 6 . The conserved amino acids were used as a rational for generating animal models of the human condition. The following constructs were generated:

TABLE 6 1 pCS2 + zf-thsd1-WT-CT2FLAG (for full rescue, positive control) 2 pCS2 + zf-thsd1-R449X-CT2FLAG (corresponding to R450X in human) 3 pCS2 + zf-thsd1-R459W-CT2FLAG (corresponding to R460W in human) 4 pCS2 + zf-thsd1-T665I-CT2FLAG (corresponding to T653I in human)

Mutations R449X, R459W and T6651 were found to be conserved between human and zebrafish. Subsequently, the nucleic acid constructs encoding these sequences were introduced into zebrafish thsd1 by PCR mutagenesis. Specifically, the thsd1 variant or the WT sequence was subcloned into pCS2+ zebrafish vector for expression. These constructs were used as tools to study cerebrovascular integrity mediated by thsd1 WT and rare variants in the zebrafish animal model selected to model a human condition.

In-Vivo Zebrafish Model

The experiment generated non-human animals carrying conserved human mutations for the study of therapies in-vivo.

While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, 6. 

1. A cell line comprised of an immortalized cell that bears a mutation in a Thrombospondin Type 1 Domain Containing 1 (THSD1) allele.
 2. The cell line of claim 1, wherein the THSD1 allele codes for an isoform of THSD1.
 3. The cell line of claim 2, wherein the isoform of THSD1 is encoded by a single codon substitution compared to the wild type allele.
 4. The cell line of claim 2, wherein the isoform of THSD1 carries a missense mutation compared to the wild type THSD1 protein.
 5. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 5. 6. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 460. 7. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 466. 8. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 600. 9. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 639. 10. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 653. 11. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele is at codon
 775. 12. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 5 encoding a Leucine for a Phenylalanine (L51-) substitution.
 13. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 460 encoding an Arginine for a Tryptophan (R460W) substitution.
 14. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 466 encoding a Glutamic Acid for a Glycine (E466G) substitution.
 15. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 600 encoding a Glycine for a Glutamic Acid (G600E) substitution.
 16. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 639 encoding a Proline for a Leucine (P639L) substitution.
 17. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 653 encoding a Threonine for a Isoleucine (T653I) substitution.
 18. The cell line of claim 3, wherein the single codon substitution in the THSD1 allele at codon 775 encoding a Serine for a Proline (S775P) substitution.
 19. The cell line of claim 1 wherein the cell line is human.
 20. The cell line of claim 1, wherein the cell line is derived from a human endothelial cell.
 21. The cell line of claim 20, wherein the human endothelial cell is derived from a brain tissue.
 22. The cell line of claim 20, wherein the human endothelial cell is derived from umbilical vein endothelial cells.
 23. The cell line of claim 20, wherein the human endothelial cell is immortalized from a primary cell. 